The phosphocreatine (PCr)/creatine kinase (CK)/ATP system is thought to be important in ATP metabolism, particularly in cells and tissues characterized by rapid and large changes in energy requirements. A creatine analogue, beta guanidinopropionic (GPA), which competitively inhibits Cr transport, has been used to study the physiological functions of PCr and CK in ATP regulation during states of energy deficit (hypoxia) and high energy turnover (seizures) in vivo using 31P NMR spectroscopy. After 3 weeks of GPA feedings, brain PCr and GPAP concentrations were about equal while CK catalyzed reaction rates decreased fourfold. Total CK activity measured analytically was two fold higher brains of GPA fed mice compared to controls. Survival during hypoxia was much greater in GPA fed mice compared to controls. Losses of brain PCr and ATP were less in GPA fed mice during both hypoxia and seizures. Both conditions produced increased CK reaction rates only in the GPA fed mice. These results strongly support a central role of guanidino compounds and the CK system in brain cellular energy regulation, in adaptation of brain ATP metabolism to critical physiological and pathological conditions such as learning and seizures or stroke, and in understanding inborn errors of energy metabolism.